help with ligation

Victor Yankiwski vy05+ at andrew.cmu.edu
Wed Apr 10 14:19:06 EST 1996


Hi,

I'm trying to ligate a variety of inserts into pCDNA3 but have not had
much luck.  The ligations are with cohesive ends, and single cuts were
treated with calf intestinal alkaline phosphatase.  On my last attempt,
I used DNA in TE at a molar ratio of 3:1 insert DNA to vector.  This
time I'm going to try a molar ratio of 10:1 insert to DNA but dont know
if I should resuspend the DNA in water before proceeding with the
ligation reaction (carried out at 25C for 1h).  Does TE inhibit the
activity of T4 ligase?

Any suggestions regarding ratios and reaction conditions would be appreciated.

Thanks
Vic
vy05 at andrew.cmu.edu



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